Age estimation is a tool for obtaining a numerical value of age for animals for which actual age is not known. Currently, age is estimated primarily from counts of growth layers deposited in several persistent tissues, primarily teeth, less often bone, and in some cases from other layered structures or from chemical signals. Growth layers in the persistent structures are similar in concept to growth rings in trees. Until use of growth layers became a feasible means of age estimation, relative measures of age, such as tooth wear, pelage or skin color, or fusion of cranial sutures, allowed individuals to be placed in age groups; these techniques largely have been replaced with methods that allow for estimation of absolute age by counting growth layers. Marine mammalogists pioneered age estimation from counting growth layers in teeth; this discovery was followed by widespread use for terrestrial mammals as well. Much of the development of this field has focused on how to ensure that age estimates are accurate and precise. That focus has been directed toward verifying the amount of time represented bv a growth layer (i.e., calibration or validation), developing increasingly better ways to prepare samples for optimizing counts, and standardizing methods to ensure that growth layer counts are consistent among studies.
Age is fundamental to interpreting and understanding many aspects of the biology of marine mammals. The traditional and most obvious use of age is for estimating parameters used in population dynamics models. Age-specific estimates of fecundity or mortality can be used in these models to project population growth, for example. Estimates of age at sexual maturation are used in absolute terms in population models, whereas changes in this parameter have been interpreted to reflect changes in population abundance or resource availability and, therefore, indicate a density-compensatory response. Population age structure would also be a useful parameter, although it is rarely known. It is possible, however, to determine the age structure of individuals removed from a population intentionally. such as through directed fisheries, or incidentally, such as bvcatch. This information then can be used to refine estimates of the impact of fisheries on those populations.
The need for accurate and precise estimates of age does not end with traditional population modeling. Of late, there has been increasing concern about the effects of contaminants on the health of marine mammals. Because many of these contaminants bioaccumulate, interpretation of the measured levels of organic or inorganic compounds must be taken as a function of the age, and reproductive condition, of the individual. Furthermore, because indices of health such as blood parameters change naturally with age, understanding the effects of contaminants or other factors on the health of individuals also requires knowing their age. With the recent epizootic events involving morbilliviruses (Tautenberger et al, 1996), the ages of individuals infected as well as those with titers indicating previous infections become important in understanding the epidemiology of these outbreaks.
I. Growth Layer Terminology
In the context of age estimation, the term growth layer is ambiguous. That is because annual increments, as a rule, comprise more than the minimum two growth layers, e.g., a broad layer and a fine layer, needed to differentiate one annual increment from the next. Other layers are usually present; these layers are often referred to as accessory or incremental layers. These are all growth layers. It has been suggested that the existence of an annual layering pattern is controlled endogenously whereas individual growth layers represent events that have a systemic effect on the animal and therefore influence the deposition of the collagen matrix or mineral in teeth or bone. Events that have been suggested include lunar cycles, maturation, pregnancy, lactation, weaning, and feeding bouts. In essence, the annual increments themselves, as well as any layers formed within the annual increments, are recording structures that reflect the physiology of an individual at the time of deposition (Klevezal, 1996). Interpreting these structures is an interesting pursuit itself and can serve as another tool for elucidating life history events for individuals. In the context of age estimation and identifying annual increments, however, they can cause errors and confusion and have resulted in semantic controversies with regard to the term “growth layer.”
To help remedy confusion in terminology, a more descriptive phrase, growth layer group (GLG), was coined at a workshop held in 1977 on estimating age in toothed whales and sire-nians ( Myrick, 1980), predominantly in reference to dentine. Its use has expanded, however, to other marine mammal species and to cement as well as dentine. A GLG is a group of layers that occur with cyclical and predictable repetition. Strictly speaking, a GLG is a generic term and does not automatically imply deposition that occurred over a 1-year period. It needs to be defined for each species and each use. For practical purposes, however, a GLG generally is defined by authors to represent 1-year’s deposition, i.e., “annual” is implied. The term “annual layer” is equivalent to “annual GLG.’
II. Calibration of Annual Layers
Verification that annual layers exist within the complement of visible layers derives from validation or calibration studies. Notably, the first confirmation of annual layers in pinniped teeth occurred soon after teeth were examined for the possibility of age estimation; Scheffer (1950) found external layers (ridges) in the canines of northern fur seals, Callorhimis ursinus, that corresponded to the known age of seals branded as pups and recovered up to 8 years later. Further studies to validate annual layering patterns and to show that patterns are consistent among individuals and species have involved three approaches: (1) examining teeth or bone from animals of known age or known history; (2) examining teeth or bone marked with tetracycline; and (3) comparing growth layers in teeth that have been removed at known intervals (multiple extractions).
For cetaceans, animals of known age or with a known history most often were captive for all or much of their lives. In the latter situation, support for annual layers then hinges on counts of the number of presumed annual layers corresponding to the known age or to the known approximate age of the animal given the length of time it spent in captivity and other data, such as its body length when removed from the wild. Initial encouragement that growth layers in dolphin teeth were annual was from three captive common bottlenose dolphins, Tursiops truncatus (Sergeant, 1959). Teeth obtained from free-ranging Tursiops of known age and known history were significant for confirming and identifying annual layering patterns and determining that annual layers in free-ranging bottlenose dolphins were similar to those in their captive conspecifics (Hohn et al. 1989). Within pinnipeds, sirenians, and sea otters (Enhydra lutris). numerous studies of free-ranging tagged or individually identified animals have compared the number of growth layers in tooth sections to known ages (e.g., Bovven et al. 1983; Arnbom et al. 1992). In many of these studies, as in cetacean studies, the actual age of individuals is greater than the “known age” because animals were captured or tagged some time after their birth. Thus, the number of growth layers counted is compared to that minimum age plus an additional number of years estimated as a function of the size of the animal at the time it was first tagged or identified. The most recent and rigorous studies counted growth layers without knowledge of the known ages of specimens in the sample, which eliminates a bias in counting. What is notable about all of these studies is that the authors concluded that they were able to identify annual growth lavers (annual GLGs) that correspond to known ages or known approximate ages of the individuals in their samples at least up to some minimum age.
True calibration of growth laver deposition over extended periods of time relative to the life span of an animal has not been attempted. To do so would require direct marking of layers, such as through administration of tetracycline, preferably at the same time each year and ideally on the animal’s birthday. Tetracycline binds permanently to actively growing mineralized tissue and fluoresces when a bone or tooth is viewed under ultraviolet light, hence its ability to serve as a marker. Two tetracycline treatments or one treatment followed by extraction have been used to unambiguously identify growth layer deposition over the period of time between the marks, providing a limited calibration; annual layers were determined from this method for several dolphin species, most extensively for spinner dolphins, Stenella longirostris (Myrick et al. 1984) and common bottlenose dolphins (Myrick and Cornell, 1990). Alternatively, multiple extractions of teeth from an individual allow for calibration but with much restricted sampling opportunities. This method has been used with free-ranging bottlenose dolphins where two teeth were extracted and growth layer deposition between extractions compared (Hohn et al. 1989). Limited opportunities exist for extensive direct calibration, although captive animals could be used for such studies as could free-ranging populations where individuals are resighted each year and could be caught, administered tetracycline, and released.
III. Tissues Commonly Used to Obtain Absolute Age Estimates
Given the importance of obtaining age estimates, various tissues and methods have been investigated for elucidating growth layers (Klevezal, 1996; McCann, 1993; Myrick, 1980). The most commonly used tissue has been teeth, as for terrestrial mammals (Klevezal and Kleinenberg, 1967). Fortunately, odontocetes (Figs. 1-3), pinnipeds, sea otters (Fig. 4), and polar bears (Ursus maritimus) have teeth that are suitable for use in estimating age. In contrast, because teeth cannot be used for baleen whales and manatees, other tissues or methods have been investigated. As alternatives, incremental layers have been found in bone, baleen, and ear plugs. Teeth have several advantages over these other tissues. The normal process of remodeling (resorption and reconstruction) in bone results in resorption of all but the most recent growth layers. For young animals, the number of bone layers may accurately reflect age; otherwise, the number of layers will be less than the age of the animal. The most useful bones are those that show negative allometry, i.e., growing more slowly than the skeleton as a whole (Klevezal, 1996), Growth layers have also been identified in baleen. Unfortunately, baleen abrades fairly quickly during normal use, and relatively few growth layers accumulate. Ear plugs are restricted to just a few species of whales and are challenging to collect.
Figure 1 Decalcified and stained midlongitudinal section in the buccal-lmgual plane from a free-ranging bottlenose dolphin known to be 3 years of age. This view shotvs only the upper half of the section. The neonatal line (NNL) represents the time when the animal was born and, therefore, is age “0″ for the purpose of estimating age. Dentine external to the neonatal line was deposited before birth and is known as prenatal dentine, whereas the neonatal line and dentine internal to it is postnatal dentine. A thin layer of enamel covered the prenatal dentine but was removed when the tooth was decalcified. The first three complete presumed annual growth layers or GLGs are marked in the sequence they were deposited. Teeth from young dolphins have very little cement and none can be seen in this photograph.
In the normal course of events, teeth do not remodel, and growth layers continue to be deposited throughout the life of the individual. Teeth are easy to collect, store, and section and have become the preferred means of age estimation for most species with teeth. Within a tooth, two tissues have been used for aging: dentine and cement. New dentine is deposited on the internal surface, i.e., from the pulp cavity side, so that layers deposited when the animal was youngest are found on the outer edges of the tooth or at the crown (Figs. 1-3). Cement or cementum wraps around the outer dentine and functions in anchoring the tooth to its alveolus. In contrast to dentine, new cemental layers are deposited on the external surface (Fig. 3). In most species of cetaceans, the cemental layer is very thin and the resulting growth layers so fine that they can be difficult to differentiate. As a result, dentine is used primarily for estimating age. Notable exceptions include the franciscana, Ponto-poria blainvillei, and the beaked whales, family Ziphiidae, where dentine is useful only for the first few years and then cement, which is extensive, must be used. In addition, for sperm whales (Physeter macrocephalus) and the beluga whale (Del-phinapterus leucas), both cement and dentine are well developed and can be used. Because cetaceans have homodont dentition (the teeth are all the same), each tooth contains the same layering pattern except for the underdeveloped teeth found most anteriorly and posteriorly in the tooth rows.
For pinnipeds, sea otters, and polar bears cement is used most frequently for age estimation (Bodkin et al, 1997; Gar-lich-Miller et al, 1993) (Fig. 4), similar to most terrestrial mammals. For many species, dentine can give accurate age estimates for young animals, but the pulp cavity either becomes occluded or the dentine deposited is too irregular to resolve additional growth layers. Notable exceptions occur in some of the phocids, such as the ringed seal (Pusa hispida), Caspian seal (Pusa caspica), and the harbor seal (Phoca vitidina), where more than 15-20 dentinal layers can be found (Stewart et al, 1996). For these species, which have heterodont dentition, canines are best for counting dentinal layers whereas postcanines are better for counting cement.
Figure 2 Decalcified and stained midlongitudinal sections of teeth from a harbor porpoise (Phocoena phocoena). Porpoise teeth are spatulate. When sectioned along the buccal-lingual plane, they appear similar to dolphin tooth sections; when sectioned sagitally, the spatulate shape is apparent. The results are comparable in both orientations for this group. A narroiv layer of cement occurs external to the dentine in the part of the tooth that was below the gum line.
Figure 3 Mandibular tooth from a sperm whale (Physeter macrocephalus) cut midlongitudinalh/ in the bticcal-lingual plane, etched in acid, and then rubbed with pencil to highlight growth layers in the dentine. Sperm whales have thick cement from which age can be estimated. In contrast to cement in the dolphin or porpoise tooth, here cement covers all of the dentine. In sperm whales and other specics with continuously growing teeth, the tooth adds layers at the bottom (root end) and pushes upward. The cement was deposited when the dentine was still below the gum line. Erupted teeth wear continuously, and in older animals the earliest deposited layers are no longer present. The ciradar structures are pulp stones that form at the edge of the pulp cavity as globular masses of secondary dentine. Pulp stones are common vn some species.
Although dentine and cement do not remodel like bone, teeth do wear down. When this occurs, it generally is not a problem for age estimation for species whose teeth show limited growth, i.e., do not continue to grow from the root but reach a maximum length when the animal is still relatively young. That is because an important marker for accurate age estimates is the neonatal line, which is deposited at birth and represents time zero for the purposes of counting growth layers (Figs. 1 and 2). As long as the neonatal line is visible, it is possible to obtain a complete count of growth layers. Initially, the neonatal line extends below the gum line. In species for which tooth growth is limited, even when the tooth wears down above the gum line, the neonatal line remains visible in the remaining tooth that was below the gum. In species with continuously growth teeth, such as the walrus (Odobenus rosmarus) (including mandibular teeth), bearded seal (Erignathus barbatus), narwhal (Monodon mono-ceros), members of the sperm whale family (Fig. 3), and the dugongs (Dugong dugon), wear continues as the tooth grows up from the root and eventually the neonatal line is worn away. When this occurs, the count of growth layers of dentine or cement is only a minimum. In some species, such as the beluga whale [Delphinapterus leucas), tooth wear is not equal and the best estimates of age are made from the least worn tooth.
Manatees (Trichechus spp.) present an unusual case for age estimation. In the related dugong, tusks (incisors) and other teeth provide a means for aging using techniques similar to those used for teeth from other species. Manatees lack tusks. Furthermore, they have an indeterminate number of molars that are constantly lost and replaced throughout life. Therefore, except in young animals, the number of growth layers in a tooth will reflect the age of the tooth but not the age of the individual manatee. As an alternative, it has been demonstrated in manatees that growth layers in tympano-periotic (auditory) bones are annual (Fig. 5) and that resorption occurs at a much slower rate than in other bones, meeting the requirement of a bone with negative allometry. More than 20 annual layers were found in many specimens and 59 found in a single animal (Mar-montel et al, 1996).
Baleen whales also present a special case for age estimation because they lack teeth. The rorquals (family Balaenopteridae) have ear plugs that are deposited in an annual layering pattern (Fig. 6) throughout life that are considered accurate for obtaining age estimates. These structures are more difficult to collect and are more fragile than teeth or bone. An advantage of ear plugs is that they do not resorb or wear. Other methods of aging have been investigated for balaenopterids, as well as other species of baleen whales. As in manatees, layers occur in the tympanic bullae (auditory) bone in bowhead (Balaena mysticetus). gray (Eschrichtius robustus), and common minke (Balaenoptera acutorostrata) whales (Christensen, 1995), often with no concomitant layers in other cranial or skeletal bones (Klevezal, 1996). Use of tympanic bullae is challenging because extensive effort is required to determine where exactly within the bullae the least amount of resorption and greatest resolution of growth layers will occur. When this region is located the maximal number of layers will be found. Otherwise, ages will be underestimated. Chemical signals, specifically amino acid racemization, have been used for dolphins and small and large species of whales (Bada et al. 1980), most recently fin (B. physalus) and bowhead whales (George et al., 1999). Age is estimated as a function of the proportion of d and l isomers of aspartic acid in the lens of the eye.
Figure 4 Growth layer deposition in cement of a knoivn-age sea otter (14 years). Images are from the same tooth section at different locations. In one location (right image), 14 ivell-defined, presumably annual layers are visible. These layers are exceptionally clear. In another location (left image), groioth layers split and merge: on the right side there appear to be fewer layers, whereas on the left side there appear to be more layers. Presumed annuli are marked on the two images, with the marks on the left image before more subjective and a particularly uncertain layer marked with a dashed line. Counts begin at the interface where the dentine meets the cement, which represents time zero for counting growth layers. Positive identification of annual layers is made by carefully following layers along the tooth to watch for splitting and merging.
IV. Collection and Preparation of Tissue for Age Estimation
When the primary tissue to be examined is dentine, especially for old animals, it is critical that a full midlongitudinal section be obtained. Otherwise, the very fine layers deposited in old animals will be missed. In toothed whales and dolphins (the odontocetes), the possibility of obtaining this midlongitudinal section is increased greatly if a tooth that is straight in the buccal-lingual plane (check to tongue) is used. Generally, the largest and straightest teeth occur near the center of the tooth row, and generally teeth are sectioned in the buccal-lingual plane. In some species, sections in the anterior-posterior plane are comparable (Fig. 2). It has become convention for studies on small odontocetes to use teeth from the center of the left ramus when possible (Myrick, 1980). When using specimens from museum collections, often the teeth will have fallen out of the alveoli and so the straightest, largest (in that priority) teeth will be optimal. For studies using cemental layers, postcanines or molars generally are the preferred tissue. In terrestrial mammals, some differences in counts of cemental growth layers among tooth sections from the same individuals have demonstrated that the thickness of the cement influences the deposition pattern, either because the cement is so narrow that layers are not readily distinguishable or because the cement is so thick that other incremental layers are apparent and may appear as annual layers (Klevezal, 1996). Differences in cemental thickness can occur both within a molar and between molars (Fig. 4). Ideally, a full investigation of the best site for sectioning can be made to select the optimal tooth and location within that tooth. When that selection has been made, midlongitudinally sections are more likely than cross sections to show all of the cemental layers, although cross sections are commonly used (Klevezal, 1996). As noted earlier , there is also variability in compact bone thickness in tympanic bullae, resulting in variability in number of growth layers visible; an investigation of the optimal site for sectioning is required. The bone is then cross-sectioned at that site. Ear plugs are sectioned centrally along the long axis of the plug.
Because growth layers are integral to bone and tooth structure, growth layer counts are not sensitive to most of the common ways of storing bones and teeth: cleaned of soft tissue and stored dry, such as in museum bone collections, or in alcohol, formalin, or glycerin. It has been suggested that long-term storage in formalin will affect growth layer counts if formalin degrades to formic acid ( Myrick, 1980). Some teeth will crack at the tip when stored dry, making sectioning a bit more difficult but not affecting the growth layers. Earplugs are stored in 5-10% buffered formalin (Lockyer, 1984). For amino acid racemization, eye lenses must be collected fresh and frozen immediately (George et al, 1999)
Figure 5 Growth layer deposition in the tympano-periotic bone of a Caribbean manatee (Trichechus manatus) that was maintained in captivity for 9 years. Eleven to 12 growth layers can be seen. These layers are primarily on the outer surface of the bone. Even at this age, the bone tissue is being resorbed and is beginning the remodeling process.
Figure 6 Ear plug from a fin whale cut midlongitudinally to expose the growth layers. The arrow denotes a significant and abrupt change in growth layer characteristics that coincides with the transition of the animal from sexually immature to sexually mature. It is called the transition phase.
Many creative methods have been tried to obtain the best resolution of growth layers (Myrick, 1980). Two of these methods have persisted and become the most widely used: untreated sections (i.e, not decalcified and stained) and decalcified and stained thin sections. The former method generally involves using a low-speed saw with diamond blades to cut a section ranging from 50 to 200 |xm thick, depending on the species and tissue, and counting layers directly from that section. The section may be mounted permanently on a microscope slide. This method was initially the most prevalent one for age estimation from teeth, a little less so for bone, and continues to be widely used because it is fast, easy, and requires little specialized equipment. The increasingly used alternative, decalcified and stained thin sections, requires additional preparation. For this method, whole teeth or thick sections from teeth or bone are decalcified in acid, sectioned on a microtome from 6 to 25 |xm, depending on the species, tissue, and microtome used, and then stained in hematoxylin and sometimes counterstained with eosin, two routine histological stains. Sections are mounted on a microscope slide. It has become increasingly evident that using the easier method produces inaccurate results for both bone and tooth sections (Stewart et al, 1996; Hohn and Fernandez, 1999). Stained thin sections allow for a much better resolution of growth layers in dentine, cement, and bone to the extent that many layers not apparent in untreated sections are visible and countable in stained sections. This difference is especially apparent in older animals where growth layers become increasingly thinner; staining is required to separate adjacent fine layers. As a result, many estimates of age using untreated sections are underestimates.
V. Consistency and Repeatability of Age Estimates
Because annual layers are not the only growth layers present, the interpretation of annual layers is often subjective. Misinterpretation of annual layers or differences in interpretation between investigators or studies lead to errors. Is one population but not another actually responding differently to exploitation or is an apparent difference simply caused by differences in age estimation? Is a population failing to recover because a growth model is incorrect or because the parameters used in that model were incorrect due to misinterpretation of annual layers?
Accuracy and precision are, in large part, influenced by the species being examined. For some species, growth layers are well defined and easily identified, whereas for other species growth layers are inherently indistinct. Annual layers in polar bear cement are notably difficult to interpret, at least during the first few years of life. Different areas in the cement have more or less distinct annual layers and accessory layers. Furthermore, different populations within a species may have different growth layer characteristics. For example, harbor porpoise from the Bay of Fundy have very distinctive growth layers, whereas those from California do not. Within studies it is common to conduct multiple readings of sections by one or more researchers to test for differences between readers or between readings for an individual reader. Measures of precision can be incorporated in models or can be used to evaluate the reliability of ages estimated for a sample.
Consistency and repeatability of age estimates can be increased if the tooth or bone sections are well prepared. Preparing these sections is a multistep process, and at each step the potential for error exists. If the end product is not well done, then the age estimate may be inaccurate or imprecise. For counts using dentine, a large source of error is using a section that is not midlongitudinal. For all sections, the incorrect stain or degree of staining (light or dark) and under- or overdecalcification also affect the final product in ways that prevent optimal resolution of all growth layers.
Even when sections are perfect, the subjective nature of counting growth layers still results in different age estimates. Descriptive models of the appearance, size, and complexity of annual layers have been developed to increase consistency, particularly between investigators. These models include photographs with the growth layers interpreted to be annual clearly marked (Hohn, 1990). Such photographs are equally valuable in individual studies to allow other investigators to determine whether the age estimates were obtained using comparable annual layering patterns. To date, such descriptive models have been prepared for bottlenose dolphins (Hohn et al, 19S9) and the franciscana (Pinedo and Hohn, 2000). Development of models for other species would increase the accuracy and precision of age estimates. Such models are particularly important and valuable when known-age specimens are available.